Preparation of crystalline forms of dihydropyrazolopyrimidinone

ABSTRACT

The instant invention relates to crystalline forms of MK-1775, an inhibitor of Weel kinase. Specifically, the instant invention relates to hemihydrates of MK-1775.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C.§371 of PCT Application No. PCT/US2010047622 filed Sep. 2, 2010, whichclaims priority from U.S. Provisional Application Ser. Nos. 61/242,428,filed Sep. 15, 2009, and 61/323,045, filed Apr. 12, 2010.

BACKGROUND OF THE INVENTION

The present invention relates to crystalline forms of2-allyl-1-[6-(1-hydroxy-1-methylethyl)pyridin-2-yl]-6-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one(Compound A) or a salt thereof, which are useful in the field oftreatment of various cancers as a kinase inhibitor, especially as a Weelkinase inhibitor.

Cells have a checkpoint mechanism such that, when the DNA therein isdamaged, then the cells temporarily stop the cell cycle and repair thedamaged DNA (Cell Proliferation, Vol. 33, pp. 261-274). In about a halfof human cancers, a cancer-suppressor gene, p53 is mutated or depletedand thereby the cells have lost the G1 checkpoint function thereof.However, such cancer cells still keep the G2 checkpoint functionremaining therein, which is considered to be one factor of lowering thesensitivity of the cells to DNA-active anticancer agents and toradiations.

A Weel kinase is a tyrosine kinase that participates in the G2checkpoint of a cell cycle. Weel phosphorylates Cdc2(Cdk1) tyrosine 15that participates in the progress to the M stage from the G2 stage in acell cycle, thereby inactivating Cdc2 and temporarily stopping the cellcycle at the G2 stage (The EMBO Journal, Vol. 12, pp. 75-85).Accordingly, in cancer cells having lost p53 function therein, it isconsidered that the G2 checkpoint function by Weel is important forrepairing the damaged DNA so as to evade the cell death. Heretofore, ithas been reported that the Weel expression reduction by RNA interferenceor the Weel inhibition by compounds may increase the sensitivity ofcancer cells to adriamycin, X ray or gamma ray (Cancer Biology &Therapy, Vol. 3, pp. 305-313; Cancer Research, Vol. 61, pp. 8211-8217).From the above, it is considered that a Weel inhibitor may inhibit theG2 checkpoint function of p53-depleted cancer cells, thereby enhancingthe sensitivity of the cells to DNA-active anticancer agents and toradiations.

Weel kinase inhibitors have been described in US Application2005/0250836, WO2003/091255, Cancer Research, Vol. 61, pp. 8211-8217, orBioorg & Med. Chem. Lett., Vol. 15, pp. 1931-1935. However, thecompounds described in these references differ structurally from thecompounds of the instant invention.

Compound A, its crystalline forms and salts thereof are described inInternational Publications WO2007/126128 (published on Nov. 8, 2007 toMerck & Co., Inc.), WO2007/126122 (published on Nov. 8, 2007 to Merck &Co., Inc.) and WO2008/133866 (published on Nov. 6, 2008, to Merck & Co.,Inc.), which are hereby incorporated by reference in their entirety.Compound A has an excellent Weel kinase inhibitory effect and is usefulin the treatment of cancer.

SUMMARY OF THE INVENTION

The instant invention relates to crystalline forms of Compound A, aninhibitor of Weel kinase. Specifically, the instant invention relates tohemihydrate Form I and hemihydrate Form II of Compound A.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characteristic X-ray diffraction pattern of the CrystallineHemihydrate Form II of Compound A.

FIG. 2 is a carbon-13 cross-polarization magic-angle spinning (CPMAS)nuclear magnetic resonance (NMR) spectrum of the crystalline HemihydrateForm II of Compound A.

FIG. 3 is a typical DSC curve of the crystalline Hemihydrate Form II ofCompound A.

FIG. 4 is a characteristic X-ray diffraction pattern of the crystallineHemihydrate Form I of Compound A.

FIG. 5 is a carbon-13 cross-polarization magic-angle spinning (CPMAS)nuclear magnetic resonance (NMR) spectrum of the crystalline HemihydrateForm I of Compound A.

FIG. 6 is a typical DSC curve of the crystalline Hemihydrate Form I ofCompound A.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel crystalline forms of Compound A, aninhibitor of Weel kinase. Compound A, also known as MK-1775, is2-allyl-1-[6-(1-hydroxy-1-methylethyl)pyridin-2-yl]-6-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one,of structural formula:

which can be prepared by procedures described in InternationalPublications WO2007/126128 and WO2007/126122, both of which published onNov. 8, 2007 to Merck & Co., Inc. Other crystalline forms and salts ofCompound A, including the monohydrate Form G and anhydrate Form B, aredescribed in International Publication WO2008/133866, which published onNov. 6, 2008, to Merck & Co., Inc., which is hereby incorporated byreference in its entirety.

The novel crystalline forms of Compound A or a salt thereof, especiallythe hemihydrates described herein, can be provided stably and constantlyfrom the standpoint of the manufacturing process, and are useful in thetreatment of cancer. The crystalline hemihydrate form I described hereinis more stable thermodynamically than other known forms at temperaturesup to 44° C. and relative humidity range of 14-93% (herein abbreviatedas “RH”). The crystalline hemihydrate form II described herein is morestable thermodynamically than other known forms at temperatures equal orhigher than 48° C. and relative humidity range of 19-93%. The relativestability of the hemihydrates and monohydrate Form G at relativehumidity higher than 93% has been difficult to establish, because thesolubility of the three crystalline forms are very close (difference <1mg/mL) and the conversion is very slow.

Also, the hemihydrate Form II can be synthesized using a crystallizationprocess that is more efficient and results in improved particle size andmorphology control when compared to other forms

The novel crystalline hemihydrate forms of Compound A, or a saltthereof, as well as Compound A per se, have a kinase-inhibitory effect,especially a Weel kinase-inhibitory effect, and are therefore useful aspharmaceutical agents for the treatment of various cancers such as braincancer, cervicocerebral cancer, esophageal cancer, thyroid cancer, smallcell cancer, non-small cell cancer, breast cancer, lung cancer, stomachcancer, gallbladder/bile duct cancer, liver cancer, pancreatic cancer,colon cancer, rectal cancer, ovarian cancer, choriocarcinoma, uterusbody cancer, uterocervical cancer, renal pelvis/ureter cancer, bladdercancer, prostate cancer, penis cancer, testicles cancer, fetal cancer,Wilms' cancer, skin cancer, malignant melanoma, neuroblastoma,osteosarcoma, Ewing's tumor, soft part sarcoma, acute leukemia, chroniclymphatic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma.

In particular, the novel crystalline forms of Compound A or a saltthereof, as well as Compound A per se, are useful as pharmaceuticalagents, for example, for the treatment of breast cancer, lung cancer,pancreatic cancer, colon cancer, ovarian cancer, acute leukemia, chroniclymphatic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma.

The term “Compound A” as referred to herein means a compound of theabove-described chemical structural formula and includes any amorphousform, polymorphic crystalline forms, hydrate, solvate and the mixturethereof.

X-ray powder diffraction studies are widely used to characterizemolecular structures, crystallinity, and polymorphism. The X-ray powderdiffraction pattern of the hemihydrate was generated on a PhilipsAnalytical X'Pert PRO X-ray Diffraction System with PW3040/60 console. APW3373/00 ceramic Cu LEF X-ray tube K-Alpha radiation was used as thesource.

The X-ray powder diffraction pattern of the Hemihydrate Form II ofCompound A is shown in FIG. 1. The Hemihydrate Form II of Compound A,also known as “the Hemihydrate Form II” or “the crystalline HemihydrateForm II of Compound A,” exhibits characteristic diffraction peakscorresponding to d-spacings of 13.81, 11.16, and 3.34 angstroms. Thehemihydrate is further characterized by the d-spacings of 6.92, 6.74,and 6.53 angstroms. The hemihydrate is even further characterized by thed-spacings of 18.17 and 4.30 angstroms.

The X-ray powder diffraction pattern of the Hemihydrate Form I ofCompound A is shown in FIG. 6. The Hemihydrate Form I of Compound A,also known as “the Hemihydrate Form 1” or “the crystalline HemihydrateForm I of Compound A,” exhibits characteristic diffraction peakscorresponding to d-spacings of 10.66, 5.30, and 4.18 angstroms. Thehemihydrate is further characterized by the d-spacings of 9.11, 8.80 and3.54 angstroms. The hemihydrate is even further characterized by thed-spacings of 18.22, 3.09 and 3.02 angstroms.

In addition to the X-ray powder diffraction patterns described above,the hemihydrates were further characterized by solid-state carbon-13nuclear magnetic resonance (NMR) spectra. The solid-state carbon-13 NMRspectra were obtained on a Bruker DSX 400WB NMR system using a Bruker 4mm H/X CPMAS probe. The carbon-13 NMR spectra utilized proton/carbon-13cross-polarization magic-angle spinning with variable-amplitude crosspolarization, and TPPM decoupling at 80 kHz. The samples were spun at10.0 kHz, and a total of 1024 scans were collected with a recycle delayof 5 seconds. A line broadening of 10 Hz was applied to the spectrabefore FT was performed. Chemical shifts are reported on the TMS scaleusing the carbonyl carbon of glycine (176.70 p.p.m.) as a secondaryreference.

The solid-state carbon-13 CPMAS NMR spectrum for the crystallineHemihydrate Form II of Compound A is shown in FIG. 2. It ischaracterized by a solid-state carbon-13 CPMAS nuclear magneticresonance spectrum showing signals at 28.6, 55.8 and 132.3 p.p.m. Thespectrum is further characterized by signals at 123.2, 118.2, and 72.3p.p.m.

The solid-state carbon-13 CPMAS NMR spectrum for the crystallineHemihydrate Form I of Compound A is shown in FIG. 5. CrystallineHemihydrate Form I is characterized by a solid-state carbon-13 CPMASnuclear magnetic resonance spectrum showing signals at 116.6, 52.4, 55.0and 31.7 p.p.m. The spectrum is further characterized by signals at145.0, 154.8, 160.1 and 169.4 p.p.m.

DSC data are acquired using TA Instruments DSC 2910 or equivalentinstrumentation. Between 1 and 7 mg sample are weighed into an open pan.This pan is then placed at the sample position in the calorimeter cell.An empty open pan is placed at the reference position. The calorimetercell is closed and a flow of nitrogen was passed through the cell. Theheating program is set to heat the sample at a heating rate of 10°C./min to a temperature of approximately 260° C. The heating program isstarted. When the run is completed, the data are analyzed using the DSCanalysis program contained in the system software. The observedendotherms and exotherms are integrated between baseline temperaturepoints that are above and below the temperature range over which theseendotherms and exotherms are observed. The data reported are the onsettemperature, peak temperature and enthalpy.

The differential calorimetry scan for the crystalline Hemihydrate FormII of Compound A is shown in FIG. 3. The DSC curve is characterized bytwo endotherms and one exotherm. The first endotherm with anextrapolated onset temperature of 134.63° C., a peak temperature of139.47° C. and enthalpy of 97.54 J/g is associated with the loss ofwater from the crystal lattice. The loss of water leads to a formationof amorphous phase, which re-crystallizes as the anhydrate Form B ofCompound A. The re-crystallization is observed in the DSC curve as anexotherm with an extrapolated onset temperature of 151.32° C., a peaktemperature of 163.52° C. and enthalpy of 30.22 J/g. The secondendotherm, with an extrapolated onset temperature of 179.74° C., a peaktemperature of 182.55° C. and enthalpy of 31.8 J/g is due to the meltingof the Anhydrate Form B of Compound A.

The differential calorimetry scan for the crystalline Hemihydrate Form Iof Compound A is shown in FIG. 8. The DSC curve is characterized by twoendotherms and one exotherm. The first endotherm with an extrapolatedonset temperature of 106.1° C. and a peak temperature of 117.9° C. isassociated with the loss of water from the crystal lattice. The loss ofwater leads to a formation of amorphous phase, which re-crystallizes asthe Anhydrate Form B of Compound A. The re-crystallization is observedin the DSC curve as an exotherm with an extrapolated peak temperature of125.1° C. The second endotherm, with an extrapolated onset temperatureof 181.3° C., a peak temperature of 182.9° C. and enthalpy of 82.33 J/gis due to the melting of the Anhydrate Form B of Compound A.

The compounds of the present invention can be prepared according to thefollowing specific examples. Those skilled in the art will readilyunderstand that known variations of the conditions and processes of thefollowing preparative procedures can be used to prepare these compounds.All temperatures are degrees Celsius unless otherwise noted.

Example 1 Preparation of Hemihydrate Form II of Compound A

Materials M.W. Amount mol eq. Compound A Monohydrate Form G 518.6 32 g0.0617 1.00 NMP (d = 1.028) 99.1 106.7 mL H₂O (d = 1.000) 18.01 319.7 mL— — 1:3 (v:v) NMP:DIW 64 mL 2:5 (v:v) EtOH:DIW 64 mL Compound AHemihydrate Form II 509.6 0.64 g 0.0013 2.04 (Seed)

To a 500 mL jacketed vessel with overhead agitation is charged 32 gCompound A Monohydrate Form G and 106.7 mL N-Methyl-2-pyrrolidone (NMP).The mixture is stirred and heated to 50° C. To this solution is added35.7 mL H₂O. The batch is then seeded with 0.64 g Compound A HemihydrateForm II. The batch is aged for 15 minutes. To this slurry is then added91 mL H₂O over 7 hours via syringe pump, followed by 193 mL over 5 hrs.The batch is then cooled from 50° C. to 20° C. over three hours. Afterthe cooldown, the batch is IKA milled (˜30 turnovers).

The batch is then filtered, washed with 64 mL 1:3 (v:v) NMP:DIW and then64 mL 2:5 (v:v) EtOH:DIW. The batch is dried in a vacuum oven at 35° C.Compound A Hemihydrate Form H (˜30.5 g) was obtained as a yellow solidin a yield of approximately 95%.

Example 2 Preparation of Hemihydrate Form II of Compound A

Raw Material Name MW Charge Amount (kg) Moles Eq/vol Compound A IPASolvate 500.95 37.8 1.00 eq (~17.5 wt % IPA, Free-Base Used as Basis)Butylated Hydroxytoluene 220.4 0.0143 0.064 150 PPM in (BHT) NMPN-Methyl-2-pyrrolidone 99.13 95.31 3.0 L/kg (NMP) freebase Deionizedwater (DIW) 18 215.3 7 L/kg freebase 3:1 DIW:NMP  ~70 L 2.25 L/kgfreebase 9:1 DIW ETOH ~150 L 4.5 L/kg freebase 9:1 DIW ETOH ~150 L 4.5L/kg freebase Compound A Hemihydrate 500.95 ~0.62 2% Seed (Form II) Seed(~1.8% water) LoadN-Methyl-2-pyrrolidone (NMP) with 150 PPM BHT (2.0 L/kg) and Compound AIPA Solvate (1.0 eq.) are charged to the reaction vessel. After allsolids have dissolved, NMP with 150 PPM BHT (1.0 L/kg) is charged torinse down the vessels/equipment. The vessel is heated to 60 C. and DIWis charged slowly to the solution until DIW content is 32 vol % DIW(˜1.3 L/kg, 32 vol % relative to NMP). The seed (˜2 wt %) is charged.The seed port is rinsed with 50 vol % NMP in DIW. The seed bed is agedfor 1 hour. Once seed bed is established, DIW is charged slowly and thewater content is brought to 50 vol % over at least 2 hours (˜1.7 L/kg).The remaining DIW is charged slowly to the batch over 2 hours. The finalwater concentration should be 70 vol % (˜3.98 L/kg, Total water addition˜7 L/kg). Terminal IKA milling at T>50° C. for ˜30 Turnovers is used toreduce particle size. If a significant number of fine particles aregenerated, a heat-cool cycle is performed. The heat cool cycle consistsof heating the batch to 80° C. over 4 hours, holding at 80° C. for 4hours, and cooling to 50° C. over 4 hours. The batch is filtered attemperature equal or higher than 50° (T≧50° C.). Displacement wash wasperformed on the batch with hot (T≧50° C.) 75% DIW:25% NMP (v:v) (2.0L/kg freebase). The displacement wash is filtered. Slurry wash of thebatch is performed with hot (T≧50° C.) 90% DIW:10:EtOH (v:v) (2.0 L/kgfreebase) and the slurry is filtered. The washing procedure was repeatedwith the same amount of hot 90% DIW:10:EtOH (v:v). To avoid cakedehydration, the batch is humid dried at 65° C.

Example 3 Preparation of Hemihydrate Form I of Compound A

Materials M.W. Amount mol eq. Compound A Hemihydrate Form II 518.6 60 g0.118 1.00 BHT 220.4 33 mg NMP (d = 1.028) 99.1 180 mL H₂O (d = 1.000)18.01 480 mL — — 2:5 (v:v) EtOH:DIW 350 mL Compound A Hemihydrate Form I509.6 1.2 g 0.0013 0.02 (Seed)

To a 2 L RB flask with overhead agitation is charged 60 g Compound AHemihydrate Form II and 180 mL N-Methyl-2-pyrrolidone (NMP). The mixtureis stirred and heated to 45° C. to dissolve. The batch is cooled to 40°C. To this solution is added 60 mL H₂O over 30 min. The batch is thenseeded with 1.2 g Compound A Hemihydrate Form I. To this slurry is thenadded 114 mL H₂O over 2.5 hours, followed by 306 mL over 2.5 hrs. Thebatch is then cooled from 40° C. to 20° C. After the cooldown, the batchis high shear rotor-stator wetmilled to reduce the particle size.

The batch is then filtered, washed with ˜350 mL 2:5 (v:v) EtOH:DIW. Thebatch is dried with vacuum and N₂ stream at room temperature (˜20° C.).Compound A hemihydrate Form I (53.1 g) was obtained as a light yellowsolid in a yield of approximately 90%.

In addition to a mixture of hemihydrates, any other crystalline form ofCompound A free base can be used as a starting material for preparationof hemihydrate Form I.

What is claimed is:
 1. Crystalline form II of2-allyl-1-[6-(1-hydroxy-1-methylethyl)pyridin-2-yl]-6-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-onehemihydrate with an X-ray powder diffraction pattern, collected usingcopper Kα radiation, corresponding to d-spacings of 13.81, 11.16, and3.34 angstroms.
 2. The crystalline hemihydrate of claim 1 which isfurther characterized by d-spacings of 6.92, 6.74, and 6.53 angstroms.3. The crystalline hemihydrate of claim 2 which is further characterizedby d-spacings of 18.17 and 4.30 angstroms.
 4. The crystallinehemihydrate of claim 1 which is further characterized by a solid-statecarbon-13 CPMAS nuclear magnetic resonance spectrum showing signals at28.6, 55.8 and 132.3 p.p.m.
 5. The crystalline hemihydrate of claim 4which is further characterized by signals at 123.2, 118.2 and 72.3p.p.m.
 6. A pharmaceutical composition comprising the crystallinehemihydrate of claim 1 and a pharmaceutically acceptable carrier.
 7. Thecrystalline hemihydrate of claim 1 which is substantially similar to theX-ray powder diffraction pattern, collected using copper Kα radiation,of FIG.
 1. 8. The crystalline hemihydrate of claim 1 which issubstantially similar to the solid-state carbon-13 CPMAS nuclearmagnetic resonance spectrum of FIG. 2.